1. Field of the Invention
The present invention relates to human monoclonal antibodies (abbreviated as MCAs hereinafter) specific for the human immunodeficiency virus, and the hybridomas which produce said MCAs. The objective of this invention is to provide human MCAs which are specific for HIV and which will be useful in the diagnosis, prevention and therapy of HIV infection.
2. Discussion of the Background
HIV is a virus which primarily infects helper T lymphocytes and brings about extreme immunological failure by destroying those cells, thereby causing AIDS (acquired immunodeficiency syndrome). In the early stage of HIV infection, some patients develop symptoms which resemble those of infectious mononucleosis, i.e., fever, fatigue, headache, etc. Subsequently, although the patient becomes asymptomatic, he/she becomes a carrier of anti-HIV antibodies in the blood. Then, after a latent period lasting a number of years, the patient develops AIDS-related complex (ARC). ARC patients exhibit various symptoms such as systemic swelling of lymph nodes, fever, general fatigue, weight loss, decreased platelet and lymphocyte levels, etc. As the disease progresses, the patient becomes susceptible to and develops Kaposi's sarcoma and various opportunistic infections such as Pneumocystis-carinii pneumonia, fungal infections, cytomegalovirusinfection, etc., which end in death. The most striking characteristics of AIDS are the decrease in helper T lymphocytes (T4), and a steady decrease in the ratio of T4 to suppressor T lymphocytes (T8), i.e., T4/T8, as the disease progresses.
AIDS was first reported in the United States of America in 1981, and it has been estimated that today there are more than 20,000 AIDS patients in the USA alone. Carriers of the virus have been estimated to number one million persons in the USA. In addition to the USA, there are also many AIDS victims in Africa and Europe, and there is a huge amount of research being carried out today on methods for the diagnosis, prevention and treatment of AIDS.
HIV, the causative agent of AIDS, is a retrovirus. This virus has been shown to be composed of RNA consisting of about 9,700 base pairs, three gag proteins (having molecular weights of 55,000, 24,000 and 17,000daltons), a reverse transcriptase (molecular weights of 66,000 and 51,000 daltons have been detected), three glycoproteins (two molecules having molecular weights of 120,000 and 41,000 daltons, and their precursor, a molecule with a molecular weight of 160,000 daltons; these glycoproteins are hereinafter abbreviated as gp120, gp41 and gp160) which comprise the envelope, and other components. Especially from the viewpoints of viral infection and the prevention thereof, the envelope, which is exposed as the surface of. HIV, carries particular importance. As a result of proteolysis, gp160 is cleaved into gp120 and gp41. Gp41 is a transmembrane protein which is incorporated into the lipid bilayer of the viral envelope, while gp120 is exposed on the outside of the envelope and some of it is released from the virus. Both gp41 and gp120 possess many sugar-binding sites, and about half of the gp120 molecule is comprised of sugars. The gp120 molecule binds to, or near to, the CD4 antigens which exist on the cell surface of helper T cells, etc., and in addition to bringing about infection of the cells by the virus, gp120 possesses activity which results in the syncytium formation in the cells.
For example, M. Robert-Guroff et al. (J. Immunol. 138: 3731, 1987) reported that the progression of the disease was slower in patients whose blood contained viral-neutratizing antibodies in comparison with patients not having such antibodies. In addition, it has been reported that the neutralizing antibodies in the blood of AIDS patients bind to gp120 (L.A. Lasky et al.: Science 233: 209, 1986; and T. J. Matthew et al.: Proc. Natl. Acad. Sci. USA 83: 9709, 1986).
Even more important there are reports of passive immunotherapy with high titer anti p-24 plasma in patients with HIV infection. This has cleared antigenemia and improved clinical prognosis. (A. Karpas et al.: Proc. Natl. Sci. USA 85: 9234, 1988; and G. G. Jackson: Lancet, 2, 647, 1988).
In light of the above background information regarding HIV and AIDS, it is obvious that neutralizing antibodies specific for viral antigens exposed on the surface of the virus or infected cells have great significance in the prevention and/or treatment of said infection.
A number of research groups have already reported successful development of mouse MCA specific for gp120. For example, T.C. Chanh et al. (Eur. J. Immunol. 16: 1465, 1986) reported that they chemically synthesized a portion of the peptide chain of gp120 and then prepared an MCA specific for that synthetic peptide. They employed that MCA in the indirect fluorescent antibody technique and reported that they were able to detect HIV infection with greater sensitivity than was possible with the reverse transcriptase determination technique. In addition, Gosting et al. (J. Clin. Microbiol.: 25, 845, 1987) reported that they solubilized HIV viral antigens, adsorbed them to a column of lentil lectin-Sepharose 4B, collected the glycoprotein fraction thereof and used it to immunize mice, and succeeded in producing anti-gp120 mouse MCA and anti-gp41 mouse MCA. Matsushita et al. (Medical Immunol. 14: 307, 1987) also reported-achieving viral neutralization with an anti-gp120 mouse MCA. These MCAs are useful in the diagnosis of HIV infection, but they are unfortunately unsuited for the tasks of prevention of HIV infection and treatment of established disease (ARC and AIDS). The reason for this is that, since those MCAs are mouse proteins, they are recognized as foreign by the human immune system if they are administered to the human body. As a result, not only would the MCA activity be inhibited by the anti-mouse MCA antibodies that would be produced by the human immune system, but anaphylactic side effects would also occur. Therefore, it is clear that, for the prevention and treatment of HIV infection in man, it is necessary to develop a human-origin MCA, not a mouse-origin MCA.
In general, human-origin anti-HIV MCAs can be produced by (1) hybridomas obtained by fusion of human B lymphocytes having the ability to produce antibodies specific for HIV and cells of established lymphoid cell lines such as myeloma cells, and (2) lymphoblastoid cells obtained by Epstein-Barr (EB) virus-induced transformation of human B lymphocytes having the ability to produce antibodies specific for HIV. From about 1980 up to the present time, much research has been carried out on the production of human MCAs, but none of those efforts have led to an established method such as in .the case of mouse MCAs because each of the approaches described above has its own special problems.
In 1987, there were two reports concerning human MCAs specific for HIV. One was by L. Evans et al. (Proceedings of the Third Congress on AIDS, TP130, 1987). They reported that they employed EB virus to transform lymphocytes from HIV-infected patients and obtained a human MCA which reacted with gag proteins having molecular weights of 55, 41 and 25 kilodaltons. That human MCA belonged to the IgG4 subclass, and it did not neutralize HIV. The second report was by B. Banapour et al. (ibid, TP114); they also employed EB virus to transform lymphocytes from anti-HIV antibody-positive subjects, fused the transformed cells with heteromyeloma cells, and obtained a human MCA which reacted with gp41. This MCA was IgG, but the subclass was not reported. This MCA also did not show HIV-neutralizing activity. Thus, in both of those reports, transformation by EB virus was employed. This technique, because it is very efficient at achieving immortalization of human B lymphocytes, is far superior to the cell fusion method. Nevertheless, the obtained lymphoblastoid cell lines produce EB virus, or even if they do not produce the virus particles, they contain the EB viral DNA which carries the potential for production of the virus. EB virus has the ability to transform lymphocytes, which means that this virus has tumorigenicity. Therefore, there is worry concerning the safety of using this EB virus transformation technique to produce a drug product for administration to humans.
It is also known that lymphoblastoid cells resulting from transformation of lymphocytes by EB virus can be further infected by HIV, and there is thus the fear that a cell line producing human MCA might be infected by both EB virus and HIV. In addition, the antibody production by lymphoblastoid cell lines presents some disadvantages in view of the facts that it is usually lower and also less stable than the level of production by hybridomas. The reason that Banapour et al. performed additional cell fusion of lymphoblastoid cell lines was to attempt to improve the antibody producing ability of those cell lines.
Accordingly, as seen above, if the immortalization of human B lymphocytes could be achieved with greater efficiency by cell fusion and if a hybridoma having the ability to produce human MCA specific for HIV could be obtained, then the resultant hybridoma would be very desirable on the basis of its having high productivity of an MCA which would moreover be safe for use as a drug.
With regard to the subclass which would be the most desirable for human MCAs, it is evident that it would be advantageous for the antibody to be of a subclass which possesses the ability to activate complement and the ability to bind to the Fc receptors on macrophages and lymphocytes. It has been demonstrated that activation of complement by the classical pathway can be achieved by the IgG1 and IgG3 subclasses, whereas IgG2 and IgG4 cannot carry out this activation (J. L. Winkelhake: Immunochem. 15: 695, 1978). Furthermore, it has also been shown that the IgG1 and IgG3 subclasses have a strong affinity for the Fc receptors of monocytes (Cosio et al.: Immuno. 44: 773, 1981). Therefore, for the objective of prevention of infection of cells, it is clear that the IgG1 and IgG3 subclasses are desirable.
However, another consideration is necessary: that of purification of the produced human MCA. Affinity chromatography using protein A can be effective for the purification of MCAs, and since IgG1 binds to protein A whereas IgG3 does not, it is clear that the IgG1 subclass of human MCA would be the most desirable subclass from the viewpoint of ease of purification.